Modulating Th2 cell levels via vMIP-I/CCR8 interaction

ABSTRACT

The present invention relates to the agonist properties of vMIP-I for the chemokine receptor CCR8 as expressed on Th2 cells. Methods of modulating a physiological signal specific to Th2 cells comprising contacting these cells with vMIP-I, agonists and antagonists thereof are disclosed. Methods for treating disease using CCR8 antagonists are also provided. The modulation of the Th2 cell populations through the vMIP-I/CCR8 interaction provides a new means for diagnosing and treating specific disease states and immunologic conditions.

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/119,033, filed Feb. 8, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates to the interaction between vMIP-Iand CCR8 receptor and the use of these molecules as well as agonists andantagonists thereof in modulating Th1 balance and the immune response.

BACKGROUND OF THE INVENTION

[0003] The chemokines are a sub-family of chemoattractant cytokines thatwere classically characterized by their ability to mediate leukocytetrafficking by binding to specific G-protein linked seven transmembranespanning receptors, or GPCRs. Chemokines are divided into four groupsbased on the primary sequence of the first two cysteines: the CXC, CC,C, and the newly discovered, CX3C families. The CXC and C families areeffective predominantly on neutrophils and lymphocytes, respectively.The CC chemokines are preferentially effective on macrophages,lymphocytes, and eosinophils.

[0004] Only about half of the chemokines have been paired to respectivereceptors. Some appear to bind to more than one receptor. The matchingof orphan receptors with the many chemokines is an ongoing process. Thematching of the ligands with receptors often provide useful insight intothe physiological functions of the individual chemokines.

[0005] The genome of the human herpesvirus 8 (HHV8), a gammaherpesviruslinked to the etiology of Kaposi's sarcoma (KS) [Moore, et al., J.Virol. 70:549-558 (1997)] encodes several chemokine-related proteinsincluding a constitutively active G-protein coupled chemokine receptor(vGPCR) that bind several CC and CXC chemokines and three viralchemokines, vMIP-I, vMIP-II and vMIP-III [Moore, et al., Science274:1739-1744 (1996); Arvanitakis, Nature 385:347-350 (1997); Nicholas,et al., J. Natl. Cancer Inst. Monogr. 23:79-88 (1998); Nicholas, et al.,Nat. Med. 3:287-292 (1997)]. In addition to its association with KS,HHV8 has also been suggested to play a role in the pathology of primaryeffusion lymphoma (PEL) [Cesarman, et al., Am. J. Pathol. 149:53-57(1996); Nador, et al., Blood 88:645-656 (1996)]. The virus has alsorecently been linked to the development of multiple myeloma [Rettig, etal., Science 276:1851-1854 (1997)]. Expression of the HHV8 GPCR inrodent fibroblasts leads to a proliferative phenotype, suggesting a rolefor this constitutively active chemokine receptor in tumorigenesis[Arvanitakis, et al., Nature 391:86-89 (1998)].

[0006] There is a need for understanding the role of thesechemokine-related proteins in HHV8 replication and the pathogenesis ofHHV8-related disease states, and generally for regimes for the treatmentof viral disease and associated pathology.

SUMMARY OF THE INVENTION

[0007] The present invention fulfills this need by providing materialsand methods for treating disease states associated with immunedysfunction. The invention is based upon the surprising discovery thatthe vMIP-I is an agonist of the chemokine receptor CCR8, a receptorlocated on activated Th2 cells.

[0008] One object of the present invention is to provide a means formodulating the balance of Th1 and Th2 cells in an animal using thevMIP/CCR8 interaction. Such modulation includes increasing anddecreasing the level of Th2 cells in order to treat various diseasestates.

[0009] Another object of the present invention provides a new method oftreating patients for various immune related disorders and diseasesusing the vMIP/CCR8 interaction.

[0010] One aspect of the invention provides a method of modulating aphysiological signal specifically to activated Th2 cells. The methodcomprises contacting activated Th2 cells with vMIP-1, or a vMIP-1 analogor antagonist. The modulating may be blocking, for example, bycontacting with a vMIP-I antagonist, e.g., an antibody. Alternatively,the modulating may be inducing with an agonist,such as, by contactingwith a CCR8 signaling ligand, e.g., vMIP-I or a vMIP-I signallinganalog. The modulating may be directing a response between a Th1 and Th2response, where the contacting is with vMIP-I, a vMIP-I antagonist orvMIP-I signaling agonist. The physiological signal may be aproliferation, apoptosis, or differentiation signal. The contacting maybe in combination with another chemokine or cytokine agonist orantagonist, including IL-12, an IL-12 antagonist, IL-1γ, or an IL-1γantagonist.

[0011] Another aspect of the invention provides methods for diagnosingand/or treating a patient infected with a virus comprising utilizing avMIP-I antagonist, such as, but not limited to, blocking monoclonalantibodies raised against vMIP-I, a modified vMIP-I peptide or a smallmolecule antagonist for vMIP-I. These entities block the interactionbetween vMIP-1 and CCR8, or prevent appropriate signaling through CCR8.This, in turn, blocks the skewing of host responses toward a Th2phenotype thus making treatment of virus-mediated tumors more effectiveand/or block anti-apoptotic effects of CCR8 again leading to moreeffective treatment of such tumors. The knowledge of the vMIP-I/CCR8interaction also makes available as treatments for viral infection otheragents that block this interaction. For example, CCR8 antagonists, I-309(natural ligand) antagonists and other CCR8 ligand antagonists can beused in the same way as a vMIP-I antagonist to block the vMIP-I/CCR8interaction.

[0012] Still another aspect of the invention provides methods forskewing an immune reponse toward a Th2 phenotype, such as, for examplein the treatment of auto-immune diseases (principally Th1 in nature) inwhich skewing to Th2 lessens disease severity and/or improves theperformance of a co-therapy, and in the treatment of bacterial/parasiteinfections, where the immune response is principally Th2 in nature. Inthe latter case, vMIP-I or signal agonists thereof increaseseffectiveness of a Th2 response leading to more rapid eradication ofthese pathogens.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 shows the results of a competition binding assay ofrecombinant I-309 or vMIP-I as assessed on CCR8-Y3 whole cells. Kd forI-309=0.65±0.17 nM (n=2) and Ki for vMIP-I=4.68±0.44 nM (n=2). Resultsare expressed as total counts vs. log concentraton of competitor.

[0014]FIG. 2 shows the chemotactic response of CCR8-Y3 cells to eithervMIP-I or I-309, as measured in the 48-well microchemotaxis assay.Results are shown as number of cells migrating/five high power (400×)fields vs. concentration of ligand. The results are representative oftwo independent experiments and each data point is the average ofduplicate wells. Vehicle alone served as a negative control.

DETAILED DESCRIPTION OF THE INVENTION

[0015] All references cited herein are incorporated in their entirety byreference.

[0016] I. General

[0017] The present invention is based, in part, upon the surprisingdiscovery that vMIP-I is an agonist of the chemokine receptor CCR8. Thehuman CCR8 receptor has been known under the names Ter1 [Napolitano etal., (1996), J. Immunol. 157:2759-2763 (1996)] ChemR1 [Samson et al.,(1996), Genomics 36:522-526], and CKR-L1 [Zabellos et al., Biochem.Biophys. Res. Commun. 846-853 (1996)] for some time. See GenBankaccession numbers AF005210 (partial) and U45983. Until recently, thedistribution of the CCR8 receptor had been characterized as lymphoidspecific, and particularly to the thymus [Napolitano et al., J.Immunol., 157:2759-2763 (1996)]. Recently, CCR8 has been shown to bepreferentially expressed on human and mouse Th2 cells. This discoverywas disclosed and claimed in a U.S. patent application Ser. No.09/190,434, the disclosure of which is hereby fully incorporated byreference. The natural human CCR8 ligand has since been shown to attractTh2-polarized T cells in vitro with considerable vigor [Zingoni et al.,J. Immunol. 161: 547-51 (1998)].

[0018] The ligand for the CCR8 chemokine receptor has been identified inhumans as the I-309 protein [Roos et al., J. Biol. Chem. 272:17251-17254(1997); Tiffany, et al, J. Exp. Med. 186:165-170 (1997); GenBankAccession Numbers M57502 and M57506], and in mouse as the TCA-3 protein[GenBank Accession Numbers M17957 and X52401]. Van Snick, et al. havereported that I-309 can block dexamethasone-mediated apoptosis of theBW5147 thymoma [Van Snick, et al., J. Immunol. 157:2570-6 (1996)],suggesting a role for CCR8 in mediating this event. vMIP-I has now beenshown to desensitize CCR8 to subsequent signaling by 1-309 and tocompete with ¹²⁵I-309 for CCR8 binding.

[0019] The activity of vMIP-I as a CCR8 agonist indicates that itsexpression by HHV8 is part of a viral program to manipulate the Th1/Th2balance of the host immune response. This finding has led to the presentinvention of vMIP-I or analogs thereof, as well as other CCR8 agonistsor antagonists, and their use as therapeutics in the treatment of viralinfections and related diseases, as well as non-virally induced diseasestates involving a Th1 or Th2 response.

[0020] The vMIP-I-CCR8 interaction also finds a role in apoptosis.vMIP-I may be utilized by viruses to prevent apoptosis of a CCR8+ cellpopulation. This may be significant to tumorigenesis in vivo or mayserve to extend the life of CCR8+ cells.

[0021] The present invention is useful in the treatment of medicalconditions which are Th1 or Th2 mediated, including, but not limited to,asthma, allergies, allergic bronchopulmonary aspergillosis, arthritis,inflammatory diseases, inflammatory bowel disease (IBD), viralinfections, various helminthic and parasitic infections, and relatedconditions.

[0022] II. Specific Binding Compositions

[0023] A. Antibodies

[0024] The present invention provides for the use of antibodies orbinding compositions which specifically bind to CCR8 or vMIP-I.Antibodies can be raised to various CCR8 or vMIP-I proteins, includingindividual, polymorphic, allelic, strain, or species variants, andfragments thereof, both in their naturally-occurring (full-length) formsor in their recombinant forms. Additionally, antibodies can be raised toCCR8 or vMIP-I proteins in both their native (or active) forms or intheir inactive, e.g., denatured forms. Anti-idiotypic antibodies mayalso be used.

[0025] A number of immunogens may be selected to produce antibodiesspecifically reactive, or selective for binding, with CCR8 or vMIP-Iproteins. Recombinant protein is a preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Naturally-occurringprotein, from appropriate sources may also be used either in pure orimpure form. Synthetic peptides may also be used as an immunogen for theproduction of antibodies to CCR8 or vMIP-I proteins. Recombinant proteincan be expressed and purified in eukaryotic or prokaryotic cells asdescribed, e.g., in Coligan, et al. (eds.) (1995 and periodicsupplements) Current Protocols in Protein Science John Wiley & Sons, NewYork, N.Y.; and Ausubel, et al (eds.) (1987 and periodic supplements)Current Protocols in Molecular Biology, Greene/Wiley, New York, N.Y.Naturally folded or denatured material can be used, as appropriate, forproducing antibodies. Either monoclonal or polyclonal antibodies may begenerated, e.g., for subsequent use in immunoassays to measure theprotein, or for immunopurification methods.

[0026] Methods of producing polyclonal antibodies are well known tothose of skill in the art. Typically, an immunogen, preferably apurified protein, is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the CCR8 protein or peptide of interest. For example,when appropriately high titers of antibody to the immunogen areobtained, usually after repeated immunizations, blood is collected fromthe animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the protein can beperformed, if desired. See, e.g., Harlow and Lane, Antibodies, ALaboratory Manual; or Coligan (ed.) Current Protocols in Immunology.Immunization can also be performed through other methods, e.g., DNAvector immunization. See, e.g., Wang, et al. Virology 228:278-284(1997).

[0027] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Typically, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell. See, Kohler and Milstein, Eur. J. Immunol.6:511-519 (1976). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods known in the art. See, e.g., Doyle, et al. (eds. 1994) andperiodic supplements) Cell and Tissue Culture: Laboratory Procedures,John Wiley and Sons, New York, N.Y. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according, e.g., to the generalprotocol outlined by Huse, et al. Science 246:1275-1281 (1989).

[0028] Antibodies or binding compositions, including binding fragmentsand single chain versions, against predetermined fragments of CCR8 orvMIP-I proteins can be raised by immunization of animals with conjugatesof the fragments with carrier proteins as described above. Monoclonalantibodies are prepared from cells secreting the desired antibody. Theseantibodies can be screened for binding to normal or defective protein.These monoclonal antibodies usually bind with at least a KD of about 1mM, more usually at least about 300 μM, typically at least about 10 μM,more typically at least about 30 μM, preferably at least about 10 μM,and more preferably at least about 3 μM or better.

[0029] In some instances, it is desirable to prepare monoclonalantibodies (mAbs) from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Antibodies: ALaboratory Manual CSH Press (1988); Goding, Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y. (1986);and particularly in Kohler and Milstein, Nature 256:495-497 (1975),which discusses one method of generating monoclonal antibodies.Summarized briefly, this method involves injecting an animal with animmunogen. The animal is then sacrificed and cells taken from itsspleen, which are then fused with myeloma cells. The result is a hybridcell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

[0030] Other suitable techniques involve selection of libraries ofantibodies in phage or similar vectors. See, e.g., Huse, et al., Science246:1275-1281 (1989); and Ward, et al. Nature 341:544-546 (1989). Thepolypeptides and antibodies of the present invention may be used with orwithout modification, including chimeric or humanized antibodies.Frequently, the polypeptides and antibodies are labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties,magnetic particles, and the like. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinantimmunoglobulins may be produced, see, Cabilly, U.S. Pat. No. 4,816,567;and Queen, et al. Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); ormade in transgenic mice, see Mendez, et al. Nature Genetics 15:146-156.(1997).

[0031] Antibody binding compounds, including binding fragments, of thisinvention can have significant diagnostic or therapeutic value. Thesemolecules can be useful as non-neutralizing binding compounds and can becoupled to toxins or radionuclides so that when the binding compoundbinds to the antigen, a cell expressing it, e.g., on its surface, iskilled. Further, these binding compounds can be conjugated to drugs orother therapeutic agents, either directly or indirectly by means of alinker, and may effect drug targeting.

[0032] B. Other Molecules

[0033] Antibodies are merely one form of specific binding compositions.Other binding compositions, which have similar uses, include moleculesthat bind with specificity to CCR8 or vMIP-I, e.g., in a partner-bindingfashion, an antibody-antigen interaction, or in a naturalphysiologically relevant protein-protein interaction, either covalent ornon-covalent, e.g., proteins which specifically associate with theprotein. The molecule may be a polymer, or chemical reagent. Afunctional analog may be a protein with structural modifications, or maybe a structurally unrelated molecule, e.g., which has a molecular shapewhich interacts with the appropriate binding determinants.

[0034] Drug screening can be performed to identify compounds havingbinding affinity to CCR8, or can block the natural interaction withligand. Subsequent biological assays can then be utilized to determineif the compound has intrinsic blocking activity and is therefore anantagonist. Likewise, a compound having intrinsic stimulating activitycan signal to the cells via the CCR8 and is thus an agonist in that itsimulates the activity of a ligand.

[0035] As indicated above, I-309, TCA-3 and vMIP-I are ligands for theCCR8 chemokine receptor [I-309: (Roos, et al., J. Biol. Chem.272:17251-17254 (1997); and Tiffany, et al., J. Exp. Med. 186:165-170(1997); GenBank M57502 and M57506), the TARC (see Imai, et al., J. Biol.Chem. 272:15036-15042 (1997); GenBank D43767); TCA-3: (GenBank M17957and X52401)]. As such, mutein antagonists of these ligands, whichmaintain receptor binding but lack signaling may be developed and usedin the practice of the invention.

[0036] Structural studies of the ligands will lead to design of newvariants, particularly analogs exhibiting agonist or antagonistproperties on the receptor. This can be combined with previouslydescribed screening methods to isolate muteins exhibiting desiredspectra of activities.

[0037] As receptor-specific binding molecules are provided, alsoincluded are small molecules identified by screening procedures. Variousligands for the receptor have been identified. In particular, it is wellknown in the art how to screen for small molecules which interfere,e.g., with ligand binding to the receptor, often by specific binding tothe receptor and blocking of binding by the natural ligand. See, e.g.,Meetings on High Throughput Screening, International BusinessCommunications, Southborough, Mass. 01772-1749. Such molecules maycompete with natural ligands, and selectively bind to the CCR8. Suchspecific binding compounds may be labeled or conjugated to toxicreagents, which are targeted to CCR8+ cells. These specific bindingreagents may similarly be used to target Th2 cells.

[0038] III. Immunoassays

[0039] Immunoassays are valuable in diagnosing a disease or disorderassociated with Th2 imbalance or pathology. Qualitative or quantitativemeasurement of a particular protein such as CCR8, which has been shownto be preferentially expressed on Th2 cells can be performed by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures in general, see Stites and Terr (eds.) Basic andClinical Immunology (7th ed.) (1991). Moreover, the immunoassays of thepresent invention can be performed in many configurations, which arereviewed extensively in Maggio, Enzyme Immunoassay CRC Press, BocaRaton, Fla. (ed. 1980); Tijan, “Practice and Theory of EnzymeImmunoassays,” Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers B. V., Amsterdam; and Harlow andLane, Antibodies, A Laboratory Manual, (1985) supra. See also Chan,Immunoassay: A Practical Guide Academic Press, Orlando, Fla. (ed. 1987);Price, Principles and Practice of Immunoassays Stockton Press, NY Newman(eds. 1991); and Ngo, Non-isotopic Immunoassays Plenum Press, NY (ed.1988).

[0040] Immunoassays for measurement of CCR8 proteins or peptides can beperformed by a variety of methods known to those skilled in the art. Inbrief, immunoassays to measure the protein can be either competitive ornoncompetitive binding assays. In competitive binding assays, the sampleto be analyzed competes with a labeled analyte for specific bindingsites on a capture agent bound to a solid surface. Preferably, thecapture agent is an antibody specifically reactive with CCR8 proteinsproduced as described above. The concentration of labeled analyte boundto the capture agent is inversely proportional to the amount of freeanalyte present in the sample.

[0041] In a competitive binding immunoassay embodiment of the invention,the CCR8 protein present in the sample competes with labeled protein forbinding to a specific binding agent, for example, an antibodyspecifically reactive with the CCR8 protein. The binding agent may bebound to a solid surface to effect separation of bound labeled proteinfrom the unbound labeled protein. Alternately, the competitive bindingassay may be conducted in liquid phase and a variety of techniques knownin the art may be used to separate the bound labeled protein from theunbound labeled protein. Following separation, the amount of boundlabeled protein is determined. The amount of protein present in thesample is inversely proportional to the amount of labeled proteinbinding.

[0042] Alternatively, a homogeneous immunoassay may be performed inwhich a separation step is not needed. In these immunoassays, the labelon the protein is altered by the binding of the protein to its specificbinding agent. This alteration in the labeled protein results in adecrease or increase in the signal emitted by label, so that measurementof the label at the end of the immunoassay allows for detection orquantitation of the protein.

[0043] CCR8 proteins may also be determined by a variety ofnoncompetitive immunoassay methods. For example, a two-site, solid phasesandwich immunoassay may be used. In this type of assay, a binding agentfor the protein, for example an antibody, is attached to a solidsupport. A second protein binding agent, which may also be an antibody,and which binds the protein at a different site, is labeled. Afterbinding at both sites on the protein has occurred, the unbound labeledbinding agent is removed and the amount of labeled binding agent boundto the solid phase is measured. The amount of labeled binding agentbound is directly proportional to the amount of protein in the sample.

[0044] Western blot analysis can be used to determine the presence ofCCR8 proteins in a sample. Electrophoresis is carried out, for example,on a tissue sample suspected of containing the protein. Followingelectrophoresis to separate the proteins, and transfer of the proteinsto a suitable solid support, e.g., a nitrocellulose filter, the solidsupport is incubated with an antibody reactive with the protein. Thisantibody may be labeled, or alternatively may be detected by subsequentincubation with a second labeled antibody that binds the primaryantibody.

[0045] The immunoassay formats described above may employ labeled assaycomponents. The label may be coupled directly or indirectly to thedesired component of the assay according to methods well-known in theart. A wide variety of labels and methods may be used. Traditionally, aradioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P was used.Non-radioactive labels include ligands which bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies which canserve as specific binding pair members for a labeled ligand. The choiceof label depends on sensitivity required, ease of conjugation with thecompound, stability requirements, and available instrumentation. For areview of various labeling or signal producing systems which may beused, see U.S. Pat. No. 4,391,904.

[0046] Antibodies reactive with a particular protein can also bemeasured by a variety of immunoassay methods. Thus modifications of theabove procedures may be used to determine the amounts or affinities ofvarious CCR8 antibodies or antibody preparation. For a review ofimmunological and immunoassay procedures applicable to the measurementof antibodies by immunoassay techniques, see Stites and Terr (eds.),Basic and Clinical Immunology (7th ed.) supra; Maggio (ed.), EnzymeImmunoassay, supra; and Harlow and Lane, Antibodies, A LaboratoryManual, supra.

[0047] Screens to evaluate the binding and activity of monoclonalantibodies and binding compositions encompass a variety of methods.Binding can be assayed by detectably labeling the antibody or bindingcomposition as described above. Cells expressing a CCR8 receptor areincubated with this antibody or binding composition, and binding isassayed by Fluorescence Activated Cell Sorting (FACS) analysis.

[0048] To evaluate Th2 depletion ability, experimental animals, e.g.,mice, are preferably induced to produce those cell types, e.g., byinfection with a parasite. Th2 cell counts are made prior to and atvarious time points after administration of a bolus of the candidatedepleting monoclonal antibody or binding composition. Levels areanalyzed in various samples, e.g., blood, serum, nasal or pulmonarylavages, or tissue biopsy staining. A successful depleting mAb orbinding composition significantly lowers the level of circulating Th2cells. Thus, a substantially pure or depleted population is at leastabout 85% pure, more preferably at least about 90% pure, and even morepreferably at least about 95, 97, or 99% pure.

[0049] Evaluation of antibodies can be performed in other animals, e.g.,humans using various methods. For example, blood samples are withdrawnfrom patients suffering from a Th2 related disease or disorder beforeand after treatment with a candidate antibody. The antibodies can beused in a diagnostic context to evaluate the extent of Th14 or Th2polarization, e.g., by FACS, tissue staining, in vitro culture.

[0050] IV. Uses

[0051] The present invention is useful in the treatment of medicalconditions or diseases associated with a Th1 or Th2 cell imbalance. See,e.g., Frank, et al., (eds.1995) Samter's Immunologic Diseases, 5th Ed.,vols. I-II, Little, Brown and Co., Boston, Mass.; Coffman, et al.,Science 245:308-310 (1989); and Frick, et al., J. Allergy Clin. Immunol.82:199-225 (1988); each of which is incorporated herein by reference.The binding specificities of the compositions described herein can. beadministered alone or in combination with another modulator of Thbalance, including, e.g., IFN-γ, IL-2, IL-4, antagonists, or othercompounds used for the treatment of symptoms, e.g., steroids such asglucocorticoids.

[0052] In particular, the selectivity of the CCR8 receptor on these celltypes of the CD4+ lineage provides a means to block the functions of therespective cell types. The expression appears on the CD4+CD8+ subset(double positive or DP). See Zinoni, et al., J.Immunol. 161:547-551(1998), incorporated herein by reference. The expression in this subsetmay correspond to cells that have already undergone positive selectionsand are destined, e.g., committed, to become CD4+ thymocytes. As I-309has been reported to prevent apoptosis in a thymoma (Van Snick, et al.,J. Immunol. 157:2570-2576 (1996)), the CCR8 may be involved in positiveselection in the double positive subset in T cell development. Ligandswhich agonize or antagonize this reaction may have importance in theregulation of apoptosis in T cell development, as well as Th2 or Th1dominant response shifting. vMIP-I or other CCR8 agonists may thereforebe used to activate, initiate, or strengthen Th2 mediated responses.Alternatively, these antagonists may block the recruitment or attractionof CCR8+ cells to the sites of ligand production, e.g., to the lung orother sites of allergic or asthmatic effects, or to block activation ormaintenance of Th2 signaling through the CCR8 receptor. In a likemanner, CD8+ cytotoxic T cells may also have a similar developmentalpathway. See O'Garra and Murphy, Curr. Opinion in Immunol. 6:458-466(1994); Palliard, et al., J. Immunol. 141:849-855 (1988); Erard, et al.,Science 260:1802-1805 (1993).

[0053] For example, CCR8 ligands, such as vMIP-I, specifically signalthe cell types expressing the receptor. Thus, it may be desirable toblock signaling to the Th2 subsets by reagents which block receptorsignaling, e.g., antibodies to receptor, antibodies to ligand, and smalldrug antagonists. The knowledge of the CCR8 mediation of Th2 traffickingand the CCR8/vMIP-I interaction provide means to block the attraction bythe CCR8 ligands.

[0054] Particular medical conditions which are Th1 or Th2 mediated, andare subject to treatment with this invention include, e.g., asthma,allergies, allergic bronchopulmonary aspergillosis, arthritis,inflammatory diseases, IBD, atopic dermatitis, viral infections, varioushelminthic and parasitic infections, and related conditions. See, e.g.,Frank, et al., Samter's Immunologic Diseases, 5th Ed., vols. I-II(eds.1995), Little, Brown and Co., Boston, Mass.; Coffman, et al.,Science 245:308-310 (1989); and Frick, et al., J. Allergy Clin. Immunol.82:199-225 (1988).

[0055] Conversely, it may be desirable to use known ligands toselectively affect specific functions mediated by Th2 cells. Thus,positive effects, e.g., blocking of apoptotic signals to these celltypes becomes feasible. There exists some evidence that I-309 can signalan anti-apoptotic signal. Thus, antagonists of the ligands may block theprotection, resulting in cell death. Conversely, blocking of attractivesignals to CCR8+ cells may be effected.

[0056] Standard immunological techniques are described, e.g., inHertzenberg, et al., Weir's Handbook of Experimental Immunology vols1-4, Blackwell Science (eds. 1996); Coligan, Current Protocols inImmunology Wiley/Greene, NY (1991); and Methods in Enzymology volumes70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. Thesetechniques are readily available in the art and allow use of thereagents for purifying cell subpopulations.

[0057] To prepare pharmaceutical or sterile compositions including theCCR8 specific binding composition, the antibody or binding compositionis admixed with a pharmaceutically acceptable carrier or excipient whichis preferably inert. Preparation of such pharmaceutical compositions isknown in the art, see, e.g., Remington's Pharmaceutical Sciences andU.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton,Pa. (1984).

[0058] Antibodies or binding compositions are normally administeredparenterally, preferably intravenously. Since such protein or peptideantagonists may be immunogenic they are preferably administered slowly,either by a conventional IV administration set or from a subcutaneousdepot, e.g. as taught by Tomasi, et al., U.S. Pat. No. 4,732,863.

[0059] When administered parenterally the antibodies or fragments areformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutically acceptable parenteralvehicle. Such vehicles are inherently nontoxic and nontherapeutic. Theantagonist may be administered in aqueous vehicles such as water, salineor buffered vehicles with or without various additives and/or dilutingagents. Alternatively, a suspension, such as a zinc suspension, can beprepared to include the peptide. Such a suspension can be useful forsubcutaneous (SQ) or intramuscular (IM) injection. The proportion ofantagonist and additive can be varied over a broad range so long as bothare present in effective amounts. The antibody is preferably formulatedin purified form substantially free of aggregates, other proteins,endotoxins, and the like, at concentrations of about 5 to 30 mg/ml,preferably 10 to 20 mg/ml. Preferably, the endotoxin levels are lessthan 2.5 EU/ml. See, e.g., Avis, et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications 2d ed., Dekker, N.Y.; Lieberman, etal., (eds.)(1990) Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker,N.Y.; Lieberman, et al., (eds.)(1990) Pharmaceutical Dosage Forms:Disperse Systems Dekker, N.Y.; Fodor, et al., Science 251:767-773(1991); Coligan (ed.), Current Protocols in Immunology; Hood, et al.,Immunology Benjamin/Cummings; Paul (ed.) Fundamental Immunology;Academic Press; Parce, et al., Science 246:243-247 (1989); Owicki, etal., Proc. Nat'l Acad. Sci. USA 87:4007-4011 (1990); and Blundell andJohnson, Protein Crystallography, Academic Press, New York (1976).

[0060] Selecting an administration regimen for an antagonist depends onseveral factors, including the serum or tissue turnover rate of theantagonist, the level of Th2 depletion, the immunogenicity of theantagonist, the accessibility of the target Th2 cells (e.g., ifnon-serum Th2 cells are to be blocked). Preferably, an administrationregimen maximizes the amount of antagonist delivered to the patientconsistent with an acceptable level of side effects. Accordingly, theamount of antagonist delivered depends in part on the particularantagonist and the severity of the condition being treated. Guidance inselecting appropriate doses is found in the literature on therapeuticuses of antibodies, e.g. Bach et al., chapter 22, in Ferrone et al.,(eds.) (1985), Handbook of Monoclonal Antibodies Noges Publications,Park Ridge, N.J.; and Russell, pgs. 303-357, and Smith et al., pgs.365-389, in Haber, et al., (eds.) (1977) Antibodies in Human Diagnosisand Therapy, Raven Press, New York, N.Y.

[0061] Determination of the appropriate dose is made by the clinician,e.g., using parameters or factors known in the art to affect treatmentor predicted to affect treatment. Generally, the dose begins with anamount somewhat less than the optimum dose and it is increased by smallincrements thereafter until the desired or optimum effect is achievedrelative to any negative side effects. Circulating activated Th2 levelsare important indicators of when an effective dose is reached.Preferably, a CCR8 antibody or binding composition thereof that is usedis derived from the same species as the animal targeted for treatment,thereby minimizing a humoral response to the reagent.

[0062] The total weekly dose ranges for antibodies or fragments thereofrange generally from about 1 ng, more generally from about 10 ng,typically from about 100 ng; more typically from about 1 μg, moretypically from about 10 μg, preferably from about 100 μg, and morepreferably from about 1 mg per kilogram body weight. Although higheramounts may be more efficacious, the lower doses typically will havefewer adverse effects. Generally the range is less than 100 mg,preferably less than about 50 mg, and more preferably less than about 25mg per kilogram body weight.

[0063] The weekly dose ranges for antagonists, e.g., antibody, bindingfragments, range from about 10 μg, preferably at least about 50 μg, andmore preferably at least about 100 μg per kilogram of body weight.Generally, the range is less than about 1000 μg, preferably less thanabout 500 μg, and more preferably less than about 100 μg per kilogram ofbody weight. Dosages are on a schedule which effects the desiredtreatment and can be periodic over shorter or longer term. In general,ranges are from at least about 10 μg to about 50 mg, preferably about100 μg to about 10 mg per kilogram body weight.

[0064] Other antagonists of the ligands, e.g., muteins, are alsocontemplated. Hourly dose ranges for muteins range from at least about10 μg, generally at least about 50 μg, typically at least about 100 mg,and preferably at least 500 mg per hour. Generally the dosage is lessthan about 100 mg, typically less than about 30 mg, preferably less thanabout 10 mg, and more preferably less than about 6 mg per hour. Generalranges are from at least about 1 μg to about 1000 μg, preferably about10 μg to about 500 μg per hour.

[0065] The present invention also provides for administration of CCR8 orligand antibodies or binding compositions in combination with knowntherapies, e.g., steroids, particularly glucocorticoids, which alleviatethe symptoms associated with excessive Th1 or Th2 responses. Dailydosages for glucocorticoids range from at least about 1 mg, generally atleast about 2 mg, and preferably at least about 5 mg per day. Generally,the dosage will be less than about 100 mg, typically less than about 50mg, preferably less than about 20 mg, and more preferably at least about10 mg per day. In general, the ranges are from at least about 1 mg toabout 100 mg, preferably from about 2 mg to 50 mg per day.

[0066] The phrase “effective amount” means an amount sufficient toameliorate a symptom or sign of the Th1 or Th2 condition. Typicalmammalian hosts include mice, rats, cats, dogs, and primates, includinghumans. An effective amount for a particular patient may vary dependingon factors such as the condition being treated, the overall health ofthe patient, the method, route, and dose of administration and theseverity of side affects. When in combination, an effective amount is inratio to a combination of components and the effect is not limited toindividual components alone.

[0067] An effective amount of antagonist decreases the symptomstypically by at least about 10%; usually by at least about 20%;preferably at least about 30%; or more preferably at least about 50%.The present invention provides reagents which find use in therapeuticapplications as described elsewhere herein, e.g., in the generaldescription for treating disorders associated with Th1/Th2 imbalances.See, e.g., Berkow (ed.), The Merck Manual of Diagnosis and Therapy,Merck & Co., Rahway, N.J.; Thorn, et al., Harrison's Principles ofInternal Medicine, McGraw-Hill, N.Y.; Gilman, et al., (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa.; Langer, Science 249:1527-1533(1990); and Merck Index, Merck & Co., Rahway, N.J.

[0068] Antibodies to CCR8 proteins may be used for the identification orsorting of cell populations expressing CCR8 protein, e.g., activated Thelper cells. Methods to sort such populations are well known in theart, see, e.g., Melamed, et al., (1990) Flow Cytometry and SortingWiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y. Populations of cellsexpressing the CCR8 receptor can also be purified using magnetic beadsas described, e.g., in Bieva, et al., Exp. Hematol. 17:914-920 (1989);Hernebtub, et al., Bioconj. Chem. 1:411-418 (1990); Vaccaro, Am.Biotechnol. Lab. 3:30 (1990).

[0069] Moreover, antisense nucleic acids may be used. For example,antisense against the ligands may function in a manner similar to ligandantagonists, and antisense against the CCR8 receptor may functionsimilar to receptor antagonists. Thus, it may be possible to blocksignaling with antisense nucleic acids. Conversely, nucleic acids forthe receptor may serve as agonists, increasing the numbers of receptorson the cell, thereby increasing cell sensitivity to ligand, and perhapsblocking the normal apoptotic signal described.

[0070] Using the assay methods described above, the antibodies orbinding compositions are useful in diagnosing disease states whichresult in Th1 or Th2 imbalances. Labeled antibodies can also be utilizedin analyzing Th2 infiltration in tissues. Antibodies raised against eachCCR8 protein are also useful to raise anti-idiotypic antibodies. Theseantibodies are useful in detecting or diagnosing various immunologicalconditions related to expression of the respective antigens.Combinations of these signals may be also pursued.

[0071] The broad scope of this invention is best understood withreference to the following examples, which are not intended to limit theinventions to the specific embodiments.

EXAMPLES

[0072] I. General Methods

[0073] Some of the standard methods are described or referenced, e.g.,in Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor Press (1982); Sambrook, et al.,Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSH Press(1989),; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, NY N.Y.; or Ausubel, et al. Current Protocols in MolecularBiology, Greene/Wiley, New York (1987) and Supplements); Innis, et al.(eds.) PCR Protocols: A Guide to Methods and Applications AcademicPress, N.Y. (1990). Methods for protein purification include suchmethods as ammonium sulfate precipitation, column chromatography,electrophoresis, centrifugation, crystallization, and others. See, e.g.,Ausubel, et al. (and periodic supplements) 1987, supra; Deutscher “Guideto Protein Purification” in Methods in Enzymology, vol. 182 (1990), andother volumes in this series; manufacturer's literature on use ofprotein purification products, e.g., Pharmacia, Piscataway, N.J., orBio-Rad, Richmond, Calif.; and Coligan, et al. (eds.) (and periodicsupplements) Current Protocols in Protein Science, John Wiley & Sons,New York, N.Y. (1995). Combination with recombinant techniques allowfusion to appropriate segments, e.g., to a FLAG sequence or anequivalent which can be fused via a protease-removable sequence. See,e.g., Hochuli, Chemische Industrie 12:69-70 (1989); Hochuli,“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98 (1990),Plenum Press, N.Y.; and Crowe, et al., QIAexpress: The High LevelExpression & Protein Purification System QIAGEN, Inc., Chatsworth,Calif. (1992).

[0074] Standard immunological techniques are described, e.g., inHertzenberg, et al. (eds.) Weir's Handbook of Experimental Immunologyvols 1-4 (1996), Blackwell Science; Coligan, Current Protocols inImmunology Wiley/Greene, NY (1991); and Methods in Enzymology volumes.70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.Methodology of cell biology techniques are described, e.g., in Celis(ed.) Cell Biology: A Laboratory Handbook Academic Press, San Diego(1998); and Doyle, et al. (eds. and periodic supplements) Cell andTissue Culture: Laboratory Procedures, John Wiley and Sons, New York,N.Y. (1994). Techniques in developmental systems are described, e.g., inMeisami (ed.), Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.), Molecular Techniques and Approaches inDevelopmental Biology Interscience.

[0075] FACS analyses are described in Melamed, et al., Flow Cytometryand Sorting Wiley-Liss, Inc., New York, N.Y. (1990); Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.,Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y. (1993).

[0076] II. Isolation of Mammalian CCR8 Encoding Sequences

[0077] The human CCR8 sequence is readily available. See, e.g., Roos, etal., J. Biol. Chem. 272:17251-17254 (1997); and Tiffany, et al., J. Exp.Med. 186:165-170 (1997); GenBank Accession Numbers AF005210 and U45983.See also WO 96/39434.

[0078] To isolate the mouse sequence, a 1057 bp fragment containing theentire ORF of the human CCR8 gene was used as probe to screen the murine129/SV genomic library in the 1/fix vector (Stratagene, La Jolla,Calif.). 350,000 phage clones were plated. Hybridization with thelabeled human CCR8 cDNA was carried out at 370 C in 50% formamide, 5×SSPE, 10× Denhardt's, 0.1% SDS, and 100 mg/ml salmon sperm DNA for 16hrs. Filters were washed three times at 50° C. in 2× SSC, 0.1% SDS, 20min. each. Positive genomic phage clones were isolated according to theplaque purification procedure. DNA was extracted, digested byrestriction endonucleases, and Southern blot analysis was performed.Hybridizing restriction fragments were subcloned in the pGem11 plasmid(Promega) and sequenced using Sequenase 2.0 (USB). DNA sequence analysiswas performed using the DNAsis/Prosis software (Hitachi).

[0079] III. Production of Cell Lines

[0080] CCR2, CCR6, CCR7 and CCR9 were stably expressed in murine BaF/3cells [Palacios, et al., Nature 391: 86-89 (1984)] using a murineretroviral sysem [Kitamura, et al., Proc. Natl. Acad. Sci. USA 92:9146-50 (1995)]. CCR8 was stably expressed in rat Y3 cells (ATCCCRL-1631) using PME-18S-neo. CCR5, XCR1, GPR9-6, and STRL33 were stablyexpressed in human embryonic kidney (HEK) 293 cells (ATCC CRL-1573)using PCDNA3.1 (Invitrogen). All lines were maintained in appropriateculture medium (RPMI or DMEM, 10% FCS, 10 ng/ml IL-3 for BaF/3 cells)containing 1 mg/ml G418 (Gibco) and were periodically tested for theirability to flux calcium in response to known ligands. Y3 cells areprepared in culture medium (RPMI of 10% FCS). Transfected cells alsocontain 1 mg/ml G418.

[0081] Other methods of evaluation of expression can also be utilized,e.g., staining and FACS analysis, tissue staining, Northern analysis,etc.

[0082] Similarly, the ligands can be recombinantly produced, purchased,or fragments synthetically produced. The ligands will be useful ingenerating further mutein antagonists or antibodies blocking theireffector functions.

[0083] IV. CCR8 Antibody Production

[0084] Appropriate mammals are immunized with appropriate amounts ofCCR8 transfected cells, e.g., intraperitoneally every 2 weeks for 8weeks. Typically, rodents are used, though other species shouldaccommodate production of selective and specific antibodies. The finalimmunization is given intravenously (IV) through the tail vein.

[0085] Generic polyclonal antibodies may be collected. Alternatively,monoclonal antibodies (“mAbs” ) can be produced. For example, four daysafter the IV injection, the spleen is removed and fused to SP2/0 and NS1cells. HAT resistant hybridomas are selected, e.g., using a protocoldesigned by Stem Cell Technologies (Vancouver, BC). After 10 days of HATselection, resistant foci are transferred to 96 well plates and expandedfor 3 days. Antibody containing supernatants are analyzed, e.g., by FACSfor binding to NIH3T3/CCR8 transfectants. Many different CCR8 mAbs aretypically produced. Those antibodies may be isolated and modified, e.g.,by labeling or other means as is standard in the art. See, e.g., Harlowand Lane, Antibodies: A Laboratory Manual CSH Press (1988); Goding,Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press,New York, N.Y. (1986). Methods to conjugate magnetic reagents, toxicentities, labels, attach the antibodies to solid substrates, to sterilefilter, etc., are known in the art.

[0086] Additionally, some of the mAbs are used to sort CCR8 positivecells in spleens from mice or primates. Various treatments of the miceare evaluated to determine the relative proportions of Th1 and Th2cells.

[0087] V. CCR8 Antagonists

[0088] Various antagonists of the CCR8 are available. For example,antibodies against the receptor itself may block the binding of ligand,thereby serving as a direct receptor antagonist. Other antagonists mayfunction by blocking the binding of ligand to receptor, e.g., by bindingto the ligand in a way to preclude the possibility of binding to thereceptor. Other antagonists, e.g., mutein antagonists, may bind to thereceptor without signaling, thereby blocking a true agonist frombinding. Many of these may serve to block the signal transmitted to theCCR8 bearing cells, specifically Th2 cells. Thus, means are provided toblock physiological signals transduced through CCR8, which isselectively expressed on Th2 cells.

[0089] In addition, the CCR8 specific reagents are useful in targetingCCR8+ cells, which are also cells. Thus, toxic conjugates selectivelydeplete CCR8+ cells from a population. This treatment is useful intreating parasitic infections, which typically involve a cellularresponse (Th1 type). The deletion of Th2 cells removes the accompanyingsuppression of Th1 effector functions.

[0090] Information on the criticality of particular residues isdetermined using standard procedures and analysis. Standard mutagenesisanalysis is performed, e.g., by generating many different variants atdetermined positions, e.g., at the positions identified above, andevaluating biological activities of the variants. This may be performedto the extent of determining positions which modify activity, or tofocus on specific positions to determine the residues which can besubstituted to either retain, block, or modulate biological activity.

[0091] Alternatively, analysis of natural variants can indicate whatpositions tolerate natural mutations. This may result from populationalanalysis of variation among individuals, or across strains or species.Samples from selected individuals are analyzed, e.g., by PCR analysisand sequencing. This allows evaluation of population polymorphisms.

Example I

[0092] This example shows identification of CCR8 as a specifichost-encoded receptor for vMIP-I.

[0093] In order to determine the host-encoded receptor(s) for vMIP-Icell lines stably transfected with known or suspected chemokinereceptors were screened for calcium flux in response to vMIP-I and otherchemokines. vMIP-I and chemokines were obtained commercially (R&DSystems, Minneapolis, Minn. or Peprotech, Rocky Hill, N.J.) or producedby DNAX/Schering-Plough.

[0094] BaF/3 cells were loaded with Fluo-3-AM (Sigma Chemical) inappropriate culture medium (RPMI or DMEM/10% serum) for 1 h at 37° C.after which cells were washed 3X in flux buffer (Hank's balanced saltsolution, 20 mM HEPES, 0.1 BSA) and aliquoted into a 96-wellblack-walled plates at a density of 1×10⁵ cells/well. HEK 293 and Y3cells were plated at a density of 5×10⁴ cells/well one day in advance ofassaying, loaded for one hour in culture medium as above and washed fourtimes. All plates were pre-coated with poly-L-lysine. Calcium flux wasmeasured in all 96-wells simultaneously and in real time using aFluorescent Imaging Plate Reader (FLIPR; Molecular Devices, Sunnyvale,Calif.) and data expressed as fluorescience units vs. time.

[0095] Y3 rat myeloid cells expressing exogenous human CCR8 (CCR8-Y3)were analyzed for reponsiveness to a variety of chemokines by measuringcalcium flux in response to ligand stimulation. The results are providedin Tables 1 and 2. TABLE 1 Chemokines which induce a calcium flux inCCR8-Y3 cells. Chemokine Response Chemokine Response MCP-1 − MIP-4 −MCP-2 − mC10 − MCP-3 − I-309 ++ MCP-4 − TECK − mMCP-5 − mTECK − Eotaxin− Mig − MIP-1a − IP-10 − MIP-1b − vMIP-I ++ DC-CK-1 − SDF-1a ++ (also innegative control) RANTES − SDF-1b ++ (also in negative control) HCC-1 −Fractalkine − (soluble domain of Fractalkine) mMIP-1g − IL-8 − 6CkinemJE − m6Ckine − GROa − BCA-1 − ENA-78 − MIP-3a − mLptn − MIP-3b − NAP-2− TARC − mGCP-2 − MDC − mLIX − mMIP-2 −

[0096] TABLE 2 vMIP-I induced calcium flux in CCR8-Y3 cells isdose-dependent. [vMIP-I] nM Relative Calcium Flux 100 ++++ 10 ++++ 1.0+++ 0.1 ++ 0.01 + 0 −

[0097] Among the various chemokines tested (all at 10 nM), only I-309and vMIP-I resulted in a calcium flux. These same cells also respondedto I-309, but not to any of thirty-nine other chemokines tested. Thecalcium response to VMIP-I was dose-dependent and observable atpicomolar concentrations. Prior incubation of CCR8-Y3 cells with vMIP-Ialso inhibited subsequent signaling to I-309 in a dose-dependent manner.See Table 3 below. Similarly, I-309 stimulation blocked subsequentsignaling to vMIP-I. See Table 4 below. The response of CCR8-Y3 cells toSDF-la and SDF-lb (Table 1) was also observed in Y3 cells transfectedwith the orphan chemokine receptor human chemokine receptor, (HCR) [Fan,et al., Biochem. Biophys. Res. Commun. 243:264-8 (1998)]/LPS induciblechemokine recepor (L-CCR) [Shimada, et al., FEBS Lett 425:490-4 (1998)]which were not responsive to either vMIP-I or I-309. Parental Y3 cellsalso responded to SDF-1a , but not vMIP-I or I-309. Twelve otherreceptors tested including CCR2, CCR3, CCR5, CCR6, CCR7, CCR9, CXCR3,CXCR4, XCR1, GPR9-6, STRL33, and HCR/L-CCR, also failed to respond tovMIP-I or I-309 with a calcium flux despite being responsive to theirknown ligands. TABLE 3 vMIP-I cross-desensitization of I-309 signalingin CCR8-Y3 cells. [vMIP-I] nM [I-309] nM Relative Calcium Flux 0  0 − 010 ++++ 0.01 10 +++ 0.1 10 ++ 1.0 10 + 10 10 +/−

[0098] TABLE 4 I-309 cross-desensitization of vMIP-I signalling inCCR8-Y3 cells. [I-309] nM [vMIP-I] nM Relative Calcium Flux 0  0 − 0 10++++ 0.01 10 +++ 0.1 10 ++ 1.0 10 + 10 10 +/−

Example II

[0099] This example provides evidence that vMIP-I desensitizessubsequent CCR8 responses to I-309 and vice versa.

[0100] The ability of vMIP-I to compete for ¹²⁵I-labeled I-309 bindingto CCR8-Y3 cells was examined. CCR8-Y3 cells were resuspended in bindingbuffer (125 mM NaCl, 25 mM HEPES, 1 mM CaCl₂, 5mM MgCl₂, 0.5% BSA, pH7.0; 200,000 cells kn 200 ml) with 0.1 nM [¹²⁵I]-I-309 (100,000 cpm).Unlabelled vMIP-I and I-309 were included as competitors whereindicated. Reactions were incubated at room temperature for three tofive hours, harvested (Unifilter-96y Harvester, Packard Instrument Co.,Meriden, Conn.) onto 96-well GF/C filter plates (Packard) and washedwith 4° C. binding buffer containing 500 mM NaCl. The filter plates weredried at room temperature overnight, scintillation cocktail(Microscint-0, Packard) was added and plates were counted (TopcountHTGS, Packard). Data was analyzed by non-linear regression (GraphPadPrism, GraphPad Software, Inc., San Diego, Calif.) and is expressed asthe average of triplicates (±standard deviation).

[0101] As shown in FIG. 1, vMIP-I compete successfully for I-309 bindingto CCR8-Y3 cells with an K_(i) of 4.68±0.44 nM which was somewhat higher(7-fold) than the K_(d) observed for I-309 binding (0.65±0.17 nM).Saturation binding of I-309 to CCR8-Y3 cells showed a K_(d) of 0.40±0.23nM (n=5). Interestingly, the EC₅₀ for CCR8-Y3 cell calcium response wasroughly equivalent for vMIP-I and I-309 stimulation (˜3.7 nM).

Example III

[0102] This example demonstrates the biological activity of vMIP-I.

[0103] In vitro chemotaxis assays were performed to determine whethervMIP-I binding to CCR8-Y3 cells could also mediate a down-streambiologic response in a 48-well microchemotaxis chamber. [Hedrick, etal., J. Immunol. 158: 1533-40 (1997)] using PVDP0-free porous membranes(5μm pore size). Assays were conducted over a one hour period and cellswere counted in an automated fashion using public domain NIH Imageanalysis software (developed at the U.S. Natl. Inst. of Health andavailable at http://rsb.info.nih.gov/nih.image). Five high power (400×)fields were counted for each of duplicate wells of a statedconcentration.

[0104] CCR8-Y3 cells were found to respond vigorously to both vMIP-I andI-309 (FIG. 2). This response showed the typical bell-shaped curvepreviously observed in microchemotaxis assays and was maximal at 10 nMfor both vMIP-I and I-309. Background migration in this assay system wasessentially zero with fewer than five cells/5 high power fieldsmigrating in response to medium alone. These data demonstrate thatvMIP-I acts as a CCR8 agonist for chemotaxis as well as calcium flux andthat vMIP-I is effective in attracting host cells in vivo.

[0105] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

What is claimed is:
 1. A method of modulating a physiological signalspecific to Th2 cells, comprising contacting said Th2 cells with vMIP-Ior an agonist or antagonist thereof.
 2. The method of claim 1, whereinsaid modulating is: a) blocking, and said contacting is with a VMIP-Iantagonist; b) inducing, and said contacting is with a VMIP-I signalingagonist; or c) directing a response between a Th1 and Th2 response, andsaid contacting is with a vMIP-I antagonist or VMIP-I signaling agonist.3. The method of claim 2, wherein said antagonist is an antibody.
 4. Themethod of claim 3, wherein said antibody binds to a vMIP-I.
 5. Themethod of claim 2, wherein said modulating is directing said response toa Th2 response, and said contacting is with said vMIP-I signalingagonist.
 6. The method of claim 1, wherein said physiological signal isa proliferation, apoptotic, differentiation, or chemoattraction signal.7. The method of claim 1, wherein said contacting is with anotherchemokine or cytokine agonist or antagonist, including IL-12, IL-12antagonist, IL-1γ, or IL-1γ antagonist.
 8. A method of treating an HHV-8viral infection in a mammal comprising administering to said mammal aneffective amount of a CCR8 antagonist or a vMIP-I antagonist.
 9. Themethod of claim 8, wherein said antagonist is an antibody.
 10. Themethod of claim 9, wherein said antibody binds to a CCR8 ligand or toCCR8.
 11. The method of claim 10, wherein said vMIP-I antagonist is avMIP-I antibody.
 12. The method of claim 10, wherein said CCR8antagonist is an I-309 antibody.
 13. The method of claim 8, wherein saidtreating is in combination with an effective amount of another chemokineor cytokine agonist or antagonist.
 14. The method of claim 13, whereinthe chemokine or cytokine agonist or antagonist is selected from thegroup consisting of IL-12, IL-12 antagonist, IL-18, or IL-18 antagonist.15. A method of treating an autoimmune disease in a mammal comprisingadministering to said mammal an effective amount of a CCR8 antagonist orvMIP-I antagonist.
 16. The method of claim 15, wherein said antagonistis an antibody.
 17. The method of claim 15, wherein said antibody bindsto a CCR8 ligand or to CCR8.
 18. A method of treating a bacterial orparasitic infection in a mammal comprising administering to said mammalan effective amount of a CCR8 agonist.
 19. The method of claim 18,wherein said CCR8 agonist is VMIP-I.
 20. The method of claim 18, whereinsaid CCR8 agonist is I-309.